1. Field of the Invention
The present invention relates to electrochemical sensors. Particularly, the present invention relates to an electrochemical sensor that is used to detect a species having a measurable vapor pressure over a sample solution by using an appropriate gas permeable membrane through which vapors diffuse. More particularly, this invention relates to an electrochemical sensor for detecting acetate in cell culture and fermentation media.
2. Description of the Prior Art
The concept of determining the partial pressure of a gas dissolved in a sample by measuring the pH of a thin film of solution separated from the sample by a gas permeable membrane was first described by Stow et al. in 1957. Thereafter, Severinghaus and Bradley constructed a probe that could measure the partial pressure of carbon dioxide (CO2) in blood. In 1973, Ross et al. described several gas sensing electrodes responding to carbon dioxide, ammonia, amines, sulfur dioxide, nitrogen dioxide, hydrogen sulfide, hydrogen cyanide, hydrogen fluoride, acetic acid, and chlorine along with their mode of operation.
Analytes of interest in the fermentation process include nutrients (e.g. glucose, glutamine), metabolites (e.g. acetate, lactate), gene regulators (e.g. phosphate, oxygen, indoleacrylic acid) and desired products (e.g. antibiotics, recombinant proteins). Many difficulties arise when analyzing fermentation media due to the fact that fermentation media are a mixture of changing concentrations of proteins, metabolites, nutrients and cells. Acetic acid is a by-product of E. coli cultures grown on glucose either in the presence of high substrate concentration or when the level of dissolved oxygen is low. Low cell yield and reduced productivity are both associated with high concentrations of acetate.
Currently, acetate is measured using several analytical methods including high-pressure liquid chromatography (HPLC), enzymatic determination and colorimetry. In colorimetry, acetate is measured by converting acetate into acetic acid and using a pH dye that undergoes color change associated with the concentration level of acetate. This is achieved by using a membrane that separates two streams from one another. On one side of the membrane is an acid donor stream containing the sample and on the other side of the membrane is a neutral receiving stream containing an acid-base indicator. The acetic acid vapors diffuse through the membrane and dissolve in the neutral receiving stream. This causes a pH drop and a color change that can be detected by absorbance at 560 nanometers. In enzymatic determination, acetate kinase is used along with the coenzyme nicotinamide adenine dinucleotide (NADH). The measurement in this analytical method is based on a change in the absorbance at 340 nanometers of the coenzyme NADH.
In xe2x80x9cPotentiometric Gas Sensing Electrodes,xe2x80x9d Ross et al., Pure and Applied Chemistry, Vol. 35, 1973, Page 473-ff, Ross et al. illustrated that it is feasible for an acetate sensor to be based on diffusion of acetic acid through a porous, gas permeable membrane. The principle used by Ross et al. is the basic Severinghaus electrode (CO2) with a different internal electrolyte and membrane material. Membrane materials used by Ross et al. are cellulose acetate, Teflon, polyvinyl chloride, polyvinyl fluoride, polypropylene, and polyethylene. A disadvantage of the Ross sensor is that the samples must be adjusted to a pH less than two. At this low pH level, interference from acidic gases such as hydrogen fluoride (HF), sulfur dioxide (SO2), formate and carbon dioxide occurs. This is so because these acidic gases, being smaller in size, have higher permeability coefficients than acetic acid. In fact, at pH 2.6 they have selectivity coefficients of more than one, thus, making the measurements of acetate concentration in fermentation impractical. The pKa of formic acid and hydrofluoric acid is 3.75 and 3.45 respectively. Consequently, despite Ross et al. suggestion, no usable and practical acetate sensors have been made for measuring cell and fermentation media.
Another problem with the prior art sensors is that they are not economical. The prior art sensors cost approximately four dollars per test not including the cost of labor to perform the test.
Therefore, what is needed is an electrochemical sensor that can detect acetate in cell culture and fermentation media. What is further needed is an acetate detecting electrochemical sensor that has a special membrane that allows for fast response time. What is still further needed is an acetate detecting electrochemical sensor that can detect sample acetate concentration when the sample is pretreated to a pH of 5.5 instead of a pH less than two. What is yet further needed is an acetate detecting electrochemical sensor with a buffer system for pretreatment of a sample that will allow for a measurable percentage of acetate to be converted into acetic acid while minimizing the effects of interfering species. What is still further needed is an acetate detecting electrochemical sensor that is more economical to use.
It is an object of the present invention to provide an electrochemical sensor that can detect acetate in cell culture and fermentation media. It is a further object of the present invention to provide an acetate detecting electrochemical sensor that has an acetic acid gas permeable membrane that allows for fast response time. It is still a further object of the present invention to provide an acetate detecting electrochemical sensor that can measure acetate concentrations within a sample when the sample is pretreated to a pH of 5.5 instead of pretreating to a pH of 2 or lower. It is yet a further object of the present invention to provide a buffer system for sample pretreatment that will allow for a measurable percentage of acetate to be converted into acetic acid while minimizing the conversion of interfering species. It is still a further object of the present invention to provide an acetate detecting electrochemical sensor that reduces the cost per test for measuring acetate concentrations in cell culture and fermentation media.
The present invention achieves these and other objectives by providing an electrochemical sensor for detecting acetate in cell culture and fermentation media. The sensor includes a pH glass electrode, an internal reference electrode, an internal reference filling solution capable of undergoing a reversible pH reaction in the presence of acetic acid, and an acetic acid gas permeable membrane.
The present invention is an acetate sensor that is based on a glass electrode with a pH sensitive tip. The glass pH electrode in combination with a reference electrode forms a complete electrochemical cell, whose e.m.f. is a function of the activity of the determinant gas in the sample. The pH sensitive tip is held against the gas permeable hydrophobic membrane so as to trap a thin film of the internal reference filling solution between the pH sensitive tip and the membrane. When the sensor is placed in a sample of determinant gas, the determinant gas diffuses through the membrane until the partial pressure of the gas in the thin film of electrolyte is equal to that in the sample. This equilibrium partial pressure of the determinant gas determines the pH of the thin film, which is measured by the glass pH electrode. For the acetate sensor, the pH of the thin film is directly proportional to the concentration of acetate in the sample.
The two basic components of the acetate sensor are an acetic acid gas permeable, hydrophobic membrane and a pH electrode. The sample is pre-treated with a buffer that maintains the sample at pH 5.5. This pretreatment of the sample to pH 5.5 is done to convert a fraction of the acetate into acetic acid and to reduce interference from formate, chloride and fluoride. At pH of 5.5, approximately fourteen percent of the acetate in the sample is converted into acetic acid. The acetic acid has a measurable vapor pressure over a sample solution so it diffuses through the membrane and dissolves in the thin film of internal reference filling solution trapped between the membrane and the pH electrode. The dissolution of the acetic acid in the internal reference filling solution causes a pH change, which is measured by the pH electrode. The magnitude of the pH change in the internal reference filling solution is a function of total acetate content in the sample.
The pH of the buffer solution resulting from the mixture of weak acid (HA) like acetic acid and its anion (Axe2x88x92) like sodium acetate is determined by the Henderson-Hasselbalch equation:                     pH        =                              pK            a                    +                      log            ⁢                          xe2x80x83                        ⁢                                          C                                  A                  -                                                            C                HA                                                                        Eq        .                  xe2x80x83                ⁢        1            
Where
Ka=the dissociation constant of the acid.
CA-=the concentration of the anion; and
CHA=the concentration of the weak acid.
Because the pKa of acetic acid is constant at 4.74 and the CA- is held constant in the internal reference filling solution, the pH of the internal reference filling solution can be related to CHA as shown in the following equation:
pH=Kxe2x88x92log CHAxe2x80x83xe2x80x83Eq. 2
where K is defined as:
K=pKa+log CAxe2x88x92xe2x80x83xe2x80x83Eq. 3
Measurement of the pH of the internal reference filling solution and relating that to the concentration of acetate in the sample forms the basis for the acetate determination.
The present invention has the advantage of pretreating samples to a pH 5.5. Prior to the present invention, samples had to be adjusted to a pH of 2 or lower to completely convert the acetate to acetic acid in sufficient quantity to provide a sufficient amount of diffusable gas for measurement. At this pH level, prior art sensors are not usable as acetate detecting sensors because of interference from acidic gases. At a pH of 5.5, approximately 14 percent of the acetate in the sample will be converted into acetic acid. This provides a good balance of acetate/acetic acid conversion to electrode sensitivity and selectivity to provide a reliable acetate detecting sensor when combined with the special membrane of the present invention.
Additional advantages and embodiments of the present invention will be set forth in part in the detailed description that follows, and in part will be apparent from the description, or may be learned by practice of the invention. It is understood that the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.